Project Summary Desmosomes are intercellular junctions that impart strength to tissues by connecting the intermediate filament networks of adherent cells. Members of two cadherin subfamilies, the desmogleins and desmocollins, mediate desmosomal adhesion in the extracellular space. While electron microscopy analyses reveal a structure with apparently high order, the arrangement and interactions of desmocollins and desmogleins that underlie the extracellular architecture of desmosomes remains unknown. This lack of knowledge has persisted in part due to the inability to produce functional desmosomal cadherin ectodomains in recombinant expression systems. Here we present preliminary data in which we present a mammalian expression system in which all seven human desmosomal cadherin ectodomains are expressed functionally and at high levels; we have determined crystal structures of ectodomains from desmogleins 2 and 3 and desmocollins 1 and 2; we have biophysically characterized binding behavior of these proteins, revealing family-wise heterophilic specificity ? where desmogleins only bind to desmocollins, and vice versa. In this proposal we will (1) perform functional mutagenesis analyses of structurally identified putative cis and trans interface regions, with biophysical and structural readout; (2) perform cell-based mutagenesis experiments to correlate molecular structural features with desmosome morphology and function in transfected cells; and (3) produce Cryo-EM tomographic reconstructions of desmosome-like junctions that spontaneously polymerize from Dsg and Dsc extracellular domains between adherent liposome membranes. Overall, this work will provide an atomic-level understanding of desmosome extracellular architecture.